The present invention is related to antisense-oligonucleotides or effective derivatives thereof hybridizing with an area of a gene coding for transforming growth factor-xcex2 (TGF-xcex2) , oligonucleotides as nonsense control nucleotides, a pharmaceutical composition comprising at least one anti-sense-oligonucleotide or effective derivatives thereof hybridizing with an area of a gene coding for TGF-xcex2 as well as a use of antisense-oligonucleotides for the manufacturing of a pharmaceutical composition for the treatment of tumors and/or the treatment of the immunosuppressive effect of TGF-xcex2.
The transforming growth factor-xcex2 (TGF-xcex2) is a factor which is, for example, secreted by human glioma cells. Human gliomas such as glioblastoma are human tumors for which at present no satisfactory therapy exists. The TGF-xcex2 supports in an autocrine manner the growing of the respective tumor cells. The factor shows immunosuppressive effects and reduces (the proliferation of such cytotoxic T-lymphocytes which otherwise would be able to destroy the glioma cells.
The supression of immune responsiveness has been well documented in patients with malignant gliomas. These patients express a variety of immunological deficiencies including cutaneous anergy, depressed antibody production, diminished numbers of circulating T-cells (Brooks, W. H., Netsky, M. G., Horwitz, D. A., Normansell, D. E. Cell mediated immunity in patients with primary brain tumors, J. Exp. Med., 136: 1931-1947, 1972 and Roszman, T., Elliott, L., Brooks, W. Modulation of T-cell function by gliomas, Immunol. Today 12: 370-374, 1991). More recent studies indicate that these impairments may result from malfunctions in physiological pathways required for normal T-cell activation and from quantitative and qualitative defects in T-cell subsets.
In Proceedings of the 82nd Annual meeting of the American Association for Cancer Research, Houston Tex., USA, May 15-18, 1991, Proc AM ASSOC CANCER RES ANNU MEET 32 (O), 1991, 427 is disclosed that factor-xcex2-antisense-oligonucleotides inhibit a human melanoma cell line under serum-enriched and stimulate under serum-free culture conditions. The results established indicate different roles of cellular TGF-xcex21 in the growth regulation of HTZ-19-cells depending on the amount of serum present in the culture medium. In addition this may indicate the biological potential and possible draw-backs of exogenously administered TGF-xcex2-antisense.
J. EXP. MED. 174 (4), 1991, 925-930, Hatzfield J. et al, xe2x80x9cRelease of early human hematopoietic progenitors from quiescene by antisense transforming growth factor xcex2-1 or Rb oligonucleotidesxe2x80x9d discloses release of early human hematopietic progenitors from quiescence by antisense transforming growth factor xcex21or Rb oligonucleotides. Rb antisense TGF-xcex2 negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.
Proceedings of the National Academy of Sciences of USA, Vo. 88, February 1991, Washington US, pages 1516-1520, Potts, J. et al., xe2x80x9cEpithelial-mesenchymal transformation of embryonic cardiac antisense oligodeoxynucleotide to transforming growth factor beta 3xe2x80x2xe2x80x9d discloses that epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor xcex23. The transformation depends on the activity of a transforming growth factor xcex2 (TGF-xcex2) molecule produced by the heart. Modified antisense oligodeoxynucleotides generated to non-conserved regions of TGF-xcex21, -2, -3 and -4 were prepared in order to examine the possible roles of these members in this transformation. As a result it has been shown that a specific member of the TGF-xcex2 family (TGF-xcex23) is essential for the epithelial-mesenchymal transformation.
WO-A 92/17206 discloses a composition for use in the treatment of wounds to inhibit scar tissue formation during healing comprising an effective activity-inhibitor amount of a growth factor neutralising agent or agents specific against only fibrotic growth factors together with a pharmaceutically acceptable carrier. The method of preparation of said composition and method of administering the composition to a host suffering from tissue wounding is also disclosed.
WO-A 90/09180 discloses methods useful in autologous bone marrow transplantation and cancer therapy. Bone marrow cells from a patient having cancer are treated with selected antisense oligonucleotides in order to deplete the bone marrow of malignant cells prior to infusion back into the bone marrow donor.
It is an object of the present invention to provide a method for the treatment of cancer cells which are correlated with an immunosuppression. Another object of the present invention is to provide an effective agent which inhibits the growth of tumor cells which are related to an immunosuppression.
According to the invention antisense-oligonucleotides or effective derivatives thereof which hybridizes with an area of gene region coding for transforming growth factor-xcex2 (TGF-xcex2) comprising the following nucleic acid sequences identified in the sequence listing under SEQ ID NO. 1-56 and 137 or comprising the following nucleic acid sequences identified in the sequence listing under SEQ ID NO. 57 to 136 each of the nucleic acids having a DNA- or RNA-type structure are able to solve the problems addressed above. Preferably, the antisense-oligonucleotides hybridize with an area of a gene region coding for growth factor-xcex21, -xcex22 and/or xcex23. The anti-sense-oligonucleotide is either able to hybridize with areas of a gene region coding for TGF-xcex2 and/or areas of a gene region coding and non coding for TGF-xcex2. For example, some nucleotides of the antisense-oligonucleotide sequence hybridizing with an area of a gene region coding for transforming growth factor-xcex2 is hybridizing with an area which does not code for the transforming growth factor whereas, the other part of the respective sequence does hybridize with a gene region coding for TGF-xcex2. Of course, it is also in the scope of the present invention that the antisense-oligo-nucleotide hybridizes with an area of a gene region just coding for growth factor-xcex2. It is also understood by the skilled person that fragments having subsequences of the antisense-oligonucleotide works according to the invention so long as production of TGF-xcex2 is reduced or inhibited.
In a preferred embodiment of the present invention the antisense-oligonucleotide or effective derivative thereof is a phosphorothioate-oligodeoxynucleotide.
According to the invention the antisense-oligonucleotides are obtainable by solid phase synthesis using phosphite triester chemistry by growing the nucleotide chain in 3xe2x80x2-5xe2x80x2 direction in that the respective nucleotide is coupled to the first nucleotide which is covalently attached to the solid phase comprising the steps of
cleaving 5xe2x80x2DMT protecting group of the previous nucleotide,
adding the respective nucleotide for chain propagation,
modifying the phosphite group subsequently cap unreacted 5xe2x80x2-hydroxyl groups and
cleaving the oligonucleotide from the solid support,
followed by working up the synthesis product.
The chemical structures of oligodeoxy-ribonucleotides are given in FIG. 1 as well as the respective structures of antisense oligo-ribonucleotides are given in FIG. 2. The oligonucleotide chain is to be understood as a detail out of a longer nucleotide chain.
In FIG. 1 lit. B means an organic base such as adenine (A), guanin (G), cytosin (C) and thymin (T) which are coupled via N9(A,G) or N1(D,T) to the desoxyribose. The sequence of the bases is the reverse complement of the genetic target sequence (mRNA-sequence). The modifications used are
1. Oligodeoxy-ribonucleotides where all R1 are substituted by
1.1 R1=O
1.2 R1=S
1.3 R1=F
1.4 R1=CH3 
1.5 R1=OEt
2. Oligodeoxy-ribonucleotides where R1 is varied at the internucleotide phosphates within one oligonucleotide 
where
B=deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence
vp=internucleotide phosphate
n=an oligodeoxy-ribonucleotide stretch of length 6-20 bases
2.1 R1a=S; R1b=O
2.2 R1a=CH3; R1b=O
2.3 R1a=S; R1b=CH3 
2.4 R1a=CH3; R1b=S
3. Oligodeoxy-ribonucleotides where R1 is alternated at the internucleotide phosphates within one oligonucleotide 
where
B=deoxy-ribonucleotide dA, dC, dG or dT depending on gene sequence
p=internucleotide phosphate
n=an oligodeoxy-ribodincleotide stretch of length 4-12 dinucleotides
3.2 R1a=S; R1b=O
3.2 R1a=CH3; R1b=O
3.3 R1a=S; R1b=CH3 
4. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at R2 with the following compounds which are covalently coupled to increase cellular uptake
4.1 cholesterol
4.2 poly(L)lysine
4.3 transferrin
5. Any of the compounds 1.1-1.5; 2.1-2.4; 3.1-3.3 coupled at R3 with the following compounds which are covalently coupled to increase cellular uptake
5.1 cholesterol
5.2 poly(L)lysine
5.3 transferrin
In the case of the RNA-oligonucleotides (FIG. 2) are the basis (adenin (A) , guanin (G), cytosin (C), uracil (U)) coupled via N9 (A,G) or N1 (c,U) to the ribose. The sequence of the basis is the reverse complement of the genetic target sequence (mRNA-sequence) . The modifications in the oligonucleotide sequence used are as follows
6. Oligo-ribonucleotides where all R1 are substituted by
6.1 R1=O
6.2 R1=S
6.3 R1=F
6.4 R1=CH3 
6.5 R1=OEt
7. Oligo-ribonucleotides where R1 is varied at the internucleotide phosphates within one oligonucleotide 
where
B=ribonucleotide dA, dC, dG or dT depending on gene sequence
p=internucleotide phosphate
n=an oligo-ribonucleotide stretch of length 4-20 bases
7.1 R1a=S; R1b=O
7.2 R1a=CH3; R1b=O
7.3 R1a=S; R1b=CH3 
7.4 R1a=CH3; R1b=S
8. Oligo-ribonucleotides where R1 is alternated at the internucleotide phosphates within one oligonucleotide 
where
B=ribonucleotide dA, dC, dG or dT depending on gene sequence
p=internucleotide phosphate
n=an oligo-ribodinucleotide stretch of length 4-12 dinucleotides
8.2 R1a=S; R1b=O
8.2 R1a=CH3; R1b=O
8.3 R1a=S; R1b=CH3 
9. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled at R2 with the following compounds which are covalently coupled to increase cellular uptake
9.1 cholesterol
9.2 poly(L)lysine
9.3 transferrin
10. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3 coupled at R3 the following compounds are covalently coupled to increase cellular uptake
10.1 cholesterol
10.2 poly(L)lysine
10.3 transferrin
11. Any of the compounds 6.1-6.5; 7.1-7.4; 8.1-8.3; 9.1-9.3; 10.1-10.3 where all R4 are substituted by
11.1 R4=O
11.2 R4=F
11.3 R4=CH3 
Modifications of the antisense-oligonucleotides are advantageous since they are not as fast destroyed by endogeneous factors when applied as this is valid for naturally occurring nucleotide sequences. However, it is understood by the skilled person that also naturally occuring nucleotides having the disclosed sequence can be used according to the invention. In a very preferred embodiment the modification is a phosphorothioat modification.
The synthesis of the oligodeoxy-nucleotide of the invention is described as an example in a greater detail as follows.
Oligodeoxy-nucleotides were synthesized by stepwise 5xe2x80x2addition of protected nucleosides using phosphite triester chemistry. The nucleotide A was introduced as 5xe2x80x2-dimethoxytrityl-deoxyadenosine(N4-benzoyl)-N,Nxe2x80x2-diisopropyl-2-cyanoethyl phosphoramidite (0.1 M); C was introduced by a 5xe2x80x2-dimethoxytrityl-deoxycytidine (N4-benzoyl)-N,Nxe2x80x2-diisopropyl-2-cyanoethyl phosphoramidite; G was introduced as 5xe2x80x2-dimethoxytrityl-deoxyguanosine(N8-isobutyryl)-N,Nxe2x80x2-diisopropyl-2-cyanoethyl phosphoramidite and the T was introduced as 5xe2x80x2-dimethodytrityl-deoxythymidine-N,Nxe2x80x2-diisopropyl-2-cyanoethyl phosphoramidite. The nucleosides were preferably applied in 0.1 M concentration dissolved in acetonitrile.
Synthesis was performed on controlled pore glass particles of approximately 150 xcexcm diameter (pore diameter 500 xc3x85) to which the most 3xe2x80x2 nucleoside is covalently attached via a long-chain alkylamin linker (average loading 30 xcexcmol/g solid support).
The solid support was loaded into a cylindrical synthesis column, capped on both ends with filters which permit adequate flow of reagents but hold back the solid synthesis support. Reagents were delivered and withdrawn from the synthesis column using positive pressure of inert gas. The nucleotides were added to the growing oligonucleotide chain in 3xe2x80x2xe2x86x925 direction. Each nucleotide was coupled using one round of the following synthesis cycle:
cleave 5xe2x80x2DMT (dimethoxytrityl) protecting group of the previous nucleotide with 3-chloroacetic acid in di chloromethane followed by washing the column with anhydrous acetonitrile. Then simultaneously one of the bases in form of their protected derivative depending on the sequence was added plus tetrazole in acetonitrile. After reaction the reaction mixture has been withdrawn and the phosphite was oxidized with a mixture of sulfur (S8) in carbon disulfid/pyridine/triethylamine. After the oxidation reaction the mixture was withdrawn and the column was washed with acetonitrile. The unreacted 5xe2x80x2-hydroxyl groups were capped with simultaneous addition of 1-methylimidazole and acetic anhydryide/lutidine/tetrahydrofuran. Thereafter, the synthesis column was washed with acetonitrile and the next cycle was started.
The work up procedure and purification of the synthesis products occured as follows.
After the addition of the last nucleotide the deoxynucleotides were cleaved from the solid support by incubation in ammonia solution. Exoxyclic base protecting groups were removed by further incubation in ammonia. Then the ammonia was evaporated under vacuum. Full-length synthesis products still bearing the 5xe2x80x2DMT protecting group were separated from shorter failure contaminants using reverse phase high performance liquid chromatography on silica C18 stationary phase. Eluents from the product peak were collected, dried under vacuum and the 5xe2x80x2-DMT protecting group cleaved by incubation in acetic acid which was evaporated thereafter under vacuum. The synthesis products were solubilized in the deionized water and extracted three times with diethylether. Then the products were dried in vacuo. Another HPLC-AX chromatography was performed and the eluents from the product peak were dialysed against excess of Trisbuffer as well as a second dialysis against deionized water. The final products were lyophilized and stored dry.
The antisense-oligonucleotides of the invention can be used as pharmaceutical composition or medicament. This medicament can be used for treating tumors in which the expression of TGF-xcex2 is of relevance for pathogenicity by inhibiting the transforming growth factor-xcex2 and thereby reducing an immunosuppression and/or inhibiting pathological angiogenesis. The reduction of immunosuppression caused by the administration of an effective dose of an antisense TGF-xcex2-oligonucleotides may be accompanied by an augmentated proliferation of cyctotoxic lymphocytes in comparison with the status before administration of the medicament. Thereupon, the lymphocytes are starting their cytotoxic activity decreasing the numbers of tumor cells.
The medicament of the present invention is further useful for the treatment of endogeneous hyperexpression of TGF-xcex2, for treatment of rest tumors, for treatment of neurofibroma, malignant glioma including glioblastoma and for the treatment and prophylaxis of skin carcinogenesis as well as treatment of esophageal and gastric carcinomas.
The effect of TGF-xcex22-specific antisense-oligonucleotides on human T cell proliferation and cytotoxicity upon stimulation with autologous cultured glioma cells was investigated. It was demonstrated that TGF-xcex22-derived phosphorothioat-derivatives S-ODN""s may specifically inhibit protein expression of TGF-xcex2 in glioma cells. In addition, TGF-xcex22-specific S-ODN""s reversxe2x80x94to a significant amountxe2x80x94immunosuppressive effects of TGF-xcex2 upon T-cell proliferation and cytotoxicity.
It has been shown that T-cell response in human brain tumor patients is clearly reduced and that tumor infiltrating lymphocytes have only marginal impact upon tumor progression of individual patients (Palma, L., Di Lorenzo, N., Guidett, B. Lymphocytes infiltrates in primary glioblastomas and recidivous gliomas, J. Neurosurg., 49: 854-861, 1978 and Ridley, A., Cavanagh, J. B. Lymphocytes infiltration in gliomas, Evidence of possible host resistance. Brain, 4: 117-124, 1971). Isolated tumor infiltrating lymphocytes from brain tumors are functionally incompetent, these immunosuppressive effects have been attributed to TGF-xcex22 in vitro and in vivo (Bodmer, S., Stromer, K., Frei, K., Siepl, Ch., de Tribolet, N., Heid, I., Fontana, A., Immunosuppression and transforming growth factor-xcex22 in glioblastoma, J. Immunol., 143: 3222-3229, 1989; Couldwell, W. T., Dore-Duffy, P., Apuzzo, M. L. J., Antel, J. P. Malignant glioma modulation of immune function: relative contribution of ifferent soluble factors, J. Neuroimmunol., 33: 89-96, 1991; Kuppner, M. C., Hamou, M. F., Sawamura, Y., Bodner, S., de Tribolet, N., Inhibition of lymphocyte function by glioblastoma derived transforming growth factor xcex22, J. Neurosurg., 71: 211-217, 1989; Maxwell, M., Galanopoulos, T., Neville-Golden, J., Antoniades, H. N., Effect of the expression of transforming growth factor-xcex22 in primary human glioblastomas on immunsuppression and loss of immune surveillance, J. Neurosurg., 76: 799-804, 1992; Palladino, M. A., Morris, R. E., Fletscher Starnes, H., Levinson, A. D., The transforming growth factor betas, A new family of immunoregulatory molecules, Ann. N.Y. Acad. Sci., 59: 181 to 187, 1990; Roszman, T., Elliott, L., Brooks, W., Modulation of T-cell function by gliomas, Immunol Today 12: 370-374, 1991).